Nanobodies targeting ABCC3 for immunotargeted applications in glioblastoma

The cancer “omics” reveal many clinically relevant alterations that are transforming the molecular characterization of glioblastomas. However, many of these findings are not yet translated into clinical practice due, in part, to the lack of non-invasive biomarkers and the limitations imposed by the blood–brain barrier. Nanobodies, camelid single-domain antibody fragments, emerge as a promising tool for immunotargeted applications for diagnosing and treating glioblastomas. Performing agnostic bioinformatic analysis from glioblastoma patient datasets, we identified ATP Binding Cassette subfamily C member 3 (ABCC3) as a suitable target for immunotargeted applications. The expression of ABCC3 is associated with poor survival and impaired response to temozolomide. Importantly, high expression of ABCC3 is restricted to glioblastoma, with negligible levels in healthy brain tissue, and further correlates with tumor grade and stemness markers. We identified three immunogenic epitopes of ABCC3 which were used to isolate nanobodies from a glioblastoma-specific phage-display nanobody library. Two nanobodies targeting ABCC3 (NbA42 and NbA213) were further characterized and demonstrated in vivo selective recognition of ABCC3 in glioblastoma xenograft mouse models upon systemic administration. We designate NbA42 and NbA213 as new candidates to implement immunotargeted applications guiding a more personalized and precise diagnosis, monitoring, and treatment of glioblastoma patients.

Glioblastoma is a highly aggressive and heterogeneous primary brain tumor. According to the WHO central nervous system (CNS) 5 classification, glioblastoma is an Isocitrate Dehydrogenase (NADP( +)) (IDH)-wildtype diffuse and astrocytic grade 4 glioma in adults which presents microvascular proliferation or necrosis or mutation of Telomerase Reverse Transcriptase (TERT) promoter or amplification of Epidermal Growth Factor Receptor (EGFR) gene or changes in + 7/ − 10 chromosome copy number 1 . Advances in molecular biology and "omic" sciences (e.g., transcriptomics, genomics, metagenomics, epigenomics, proteomics, metabolomics) provide clinically relevant biomarkers for the further development of powerful tools to study the biology of glioblastoma and to identify novel therapies 2 .
Despite advances in the molecular classification of glioblastoma, there is an urgent need to designate novel molecular targets to improve patient management strategies. Overall survival has not changed in past decades and remains at 14.6 months for patients with primary glioblastoma. This life expectancy is reduced to 6.7 months after relapse 3,4 . The current standard of care of glioblastoma patients includes maximal surgical resection, followed by radiotherapy with concomitantly and adjuvant chemotherapy with the alkylating agent temozolomide (TMZ) 3,4 . TMZ is an alkylating prodrug that delivers a methyl group to purine bases of DNA generates O 6 -methylguanine in DNA, a cytotoxic lesion removed by O 6 -methylguanine DNA methyltransferase (MGMT). Methylation of the MGMT promoter results in gene silencing and correlates with better outcome in TMZ-treated glioblastoma patients 5 . Although a tissue sample from surgery or biopsies is often required, genetic alterations and epigenetic modifications have uncovered their relevance towards more personalized treatment strategies. The combination of IDH1 mutations and MGMT promoter methylation status offers a higher predictive potential of survival in glioblastoma patients than either alone 6 .

Results
ABCC3 is a target for glioblastoma immunotargeted applications. Candidate targets of glioblastoma for the development of immunotargeted applications were selected based on (1) their higher gene expression on glioblastoma compared with healthy tissue, and (2) the location of the encoded protein on the extracellular surface of the plasma membrane. To identify the most differentially expressed genes in glioblastoma, we performed a bioinformatic analysis of 142 glioblastoma and 5 normal brain samples (RNA-Seq) deposited in The Cancer Genome Atlas (TCGA) 26 using limma (v. 3.24.15, R package) 27 . According to these criteria, 287 genes (Supplementary Table S1) were identified and ranked by differential expression (Log 2 FC). 9 genes were identified to be the most differentially expressed in glioblastoma compared to healthy tissue (Log 2 FC > 4) encoding for membrane-associated proteins (Fig. 1a). Further analysis using GlioVis 28 revealed that only the expression of ABCC3 and CA9 correlated with worse patient overall survival (Fig. 1b, and Supplementary Figs. S1a, S1d, S2). The role of CA9 in glioblastoma has been described 29 and nanobodies against CA9 have been previously developed 30 . In addition, the implication of ABCC3 in drug resistance and stemness potential of a variety of tumors has been described 20 . Therefore, we further investigated the role of ABCC3 in glioblastoma.
Despite studies that have suggested higher ABCC3 expression levels in MGMT methylated patient samples 20 , we found similar ABCC3 levels of expression independently of the MGMT methylation status (Fig. 1c). Importantly, we found that higher expression of ABCC3 in patients with methylated MGMT promoter correlates with a worse prognosis (Fig. 1d). A possible role for ABCC3 in TMZ resistance and cancer stemness could be hypothesized in glioblastoma. Importantly, we found a correlation between the expression of stemness markers (CD44 and FUT4 (CD15)) with ABCC3 in glioblastoma (Fig. 1e, and Supplementary Fig. S1c,d,h,i). ABCC3 presents lower expression levels in normal brain than in glioblastoma samples (Fig. 1f, and Supplementary Fig. S3), and its expression increases with glioma tumor grade (Figs. 1g, and Supplementary Fig. S1b,e). ABCC3 is located on human chromosome 17 and its expression in glioblastoma is independent of copy number alterations (Fig. 1h) suggesting that ABCC3 expression is a regulated process during glioblastoma development instead of a consequence of chromosomal instability (e.g., duplication, deletion, or loci amplification). We also observe high levels of expression of ABCC3 in a panel of glioblastoma cell lines (Fig. 1i). These data point to ABCC3 as a suitable molecular target that meets the requirements for further developing nanobody-based immunotargeted tools for glioblastoma.  25 . Screening and selection of nanobodies targeting ABCC3 were performed following a peptide-based strategy. We predicted immunogenic epitopes within the amino acidic sequence of ABCC3 (UniProtKB reference: O15438) using BepiPred-2.0 analy-  www.nature.com/scientificreports/ sis software 31 . Candidate immunogenic epitopes were selected based on (1) their sequential nature, as linear B-cell epitopes are commonly used for immunizations and antibody production 32 , (2) their length, with limitation to a minimum of 11 and a maximum of 30 amino acids to approximate the size of current linear epitopes 33,34 , and (3) their outer surface location at the plasma membrane, restricted to extracellular regions of the Membrane Spanning Domains (MSDs) and N-terminus of ABCC3 (Fig. 2a). The bioinformatic analysis yielded 57 potential immunogenic epitopes (Supplementary Table S2), from which four peptides met these requirements. Their precise alignment with the ABCC3 sequence was confirmed utilizing the Basic Local Alignment Search Tool (BLAST) Protein (BlastP). As shown in Fig. 2a, one of the selected peptides (ABCC3-A, amino acids 3-32) is located in the N-terminus, while the other three (ABCC3-K, amino acids 160-170; ABCC3-V, amino acids 557-569; and ABCC3-L, amino acids 993-1010) are located within extracellular loops of MSDs of ABCC3. Three of them were synthesized with an addition of a 6× His-tag at the C-terminus (ABCC3-A and ABCC3-K) or N-terminus (ABCC3-L), for further screening of nanobodies. . Extracellular N-glycosylation sites may regulate substrate specificity, stability, and localization of ABCC3 19 . The location and amino acid sequence of the selected peptides in extracellular regions utilized to isolate specific nanobodies targeting ABCC3 from a phage display library is shown. (b) Amino acid sequence of the isolated nanobodies targeting ABCC3 depicting their Framework Regions (FR1-4, grey) and Complementarity Determining Regions (CDR1, pink; CDR2, green; and CDR3, given in alphabetic order, blue). Nanobodies NbA49, NbK2, NbK39, NbK229 and NbL115 present a VH-like VHH imprint in their framework 2 region (GLEW sequence). Nanobody NbL51 presents an extremely short CDR3 domain, which the longer CDR1 loop might compensate for, and a modified GLEW motif with a three-amino acid insertion. Some nanobodies targeting ABCC3 have an R118 residue instead of W118 as the first amino acid of the framework 4 region, which is exclusive of VHHs and not presented in humans, mouse nor camel VH. (c) Screening of the immunoreactivity of nanobodies targeting ABCC3 by flow cytometry. The immunoreactivity of 2 nanobodies targeting peptide ABCC3-A (NbA42, NbA213), 2 nanobodies targeting peptide ABCC3-K (NbK2, NbK39), and 1 nanobody targeting peptide ABCC3-L (NbL51) was confirmed. A549 cell line (A549 WT) and its ABCC3 knocked-out derivative cell line (A549 ABCC3KO ) were used as positive and negative controls. Secondary staining controls are also shown. www.nature.com/scientificreports/ Identification and screening of nanobodies targeting ABCC3. For nanobody enrichment, each ABCC3-derived peptide was used for three rounds of biopanning of the phage-display library 25 . We identified 12 nanobodies with different and complete sequences ( Fig. 2b; Supplementary Table S3). We selected five nanobodies recognizing peptide ABCC3-A (NbA42, NbA49, NbA92, NbA213, NbA237), four nanobodies with ABCC3-K (NbK2, NbK39, NbK145, NbK229), and three nanobodies with ABCC3-L (NbL51, NbL91, NbL115). The selective targeting of the isolated nanobodies against ABCC3 was evaluated by flow cytometry (Supplementary Figure S4a). The human lung adenocarcinoma A549 cell line (A549 WT) presents the highest levels of ABCC3 expression (Human Protein Atlas, http:// www. prote inatl as. org) 35 and was selected as a positive control for the initial experiments. We knocked-out ABCC3 by CRISPR/Cas9 technology. The resulting cell line (A549 ABCC3KO ) was used as a negative control ( Supplementary Fig. S4b). Five nanobodies showed significant differential binding capacity (Fig. 2c). The immunoreactivity of two nanobodies targeting peptide ABCC3-A (NbA42, NbA213), two nanobodies targeting peptide ABCC3-K (NbK2, NbK39), and one nanobody targeting peptide ABCC3-L (NbL51) was confirmed. NbA42 and NbA213 were the nanobodies with the highest potential to detect ABCC3 in vitro and were further evaluated.
Molecular characterization of anti-ABCC3 nanobodies. Nanobodies NbA42 and NbA213 specifically recognize ABCC3. The potential of targeting ABCC3 by NbA42 and NbA213 was evaluated by flow cytometry using ABCC3-expressing and loss-of-function control cell lines (Fig. 3a), demonstrating the potential to discriminate different proportions of these cell types in vitro. Subsequently, both nanobodies were labeled with fluorescein-5-isothiocyanate (FITC) to determine their equilibrium dissociation constant (K D ). The affinity of NbA42 and NbA213 was assessed by targeting the control cell line A549 WT, using A549 ABCC3KO as a nonspecific background control (Fig. 3b). Results showed that both nanobodies bind whole-length ABCC3 protein in vitro (K D = 115.3 ± 89.95 µM for NbA42 and K D = 52.58 ± 25.79 µM for NbA213). We confirmed the specific recognition of NbA42 and NbA213 to peptide ABCC3-A among the other ABCC3-derived peptides (e.g., AB-CC3-K, and ABCC3-L) by indirect ELISA (Fig. 3c). The combination of NbA42 and NbA213 demonstrated no synergy to detect ABCC3 in vitro (Fig. 3d), further validating the common recognition of peptide ABCC3-A.
NbA42 and NbA213 can target ABCC3 in vivo. The capacity of NbA42 and NbA213 to target ABCC3 in vivo was evaluated in xenografted tumor-bearing mice of the A549 and A549 ABCC3KO control cell lines. Nanobodies were administered intraperitoneally (i.p.) at 15 mg·kg −1 and their specific binding was analyzed 1 h after systemic injection (Fig. 4a). Similar to the in vitro data, systemic injection of NbA42 and NbA213 demonstrated selective detection of ABCC3 in vivo (Fig. 4b). Both nanobodies specifically bound A549 WT xenografted tumors (ABCC3-expressing cells), compared with the background of A549 ABCC3KO xenografted tumors (ABCC3-loss-offunction cells). NbA42 and NbA213 showed minimal residual signals in other tissues ( Supplementary Fig. S5a). Accordingly to previously reported data on the pharmacokinetic profile of other nanobodies [36][37][38] NbA42 and NbA213 were early detected in blood plasma after i.p. administration and cleared from circulation by renal clearance (Supplementary Fig. S5b). NbA42 and NbA213 were not detectable in plasma samples 4 h upon systemic administration.
NbA42 and NbA213 also target ABCC3-expressing glioblastoma cell lines. Both nanobodies showed a similar in vitro targeting of ABCC3 in U-87 MG, U251 and T98G cells ( Supplementary Fig. S6). We next evaluated the capacity of NbA42 and NbA213 to selectively target glioblastoma cells in vivo. First, we analyzed the selective targeting of NbA42 and NbA213 in U-87 MG and T98G heterotopic xenografted tumor-bearing mice in vivo (Fig. 4c). One hour after systemic administration, both nanobodies recognized ABCC3-expressing heterotopic xenografted glioblastoma tumors. We next evaluated the potential of NbA42 and NbA213 to detect ABCC3expressing orthotopic brain tumors (Fig. 5a). Two orthotopic glioblastoma mouse models, with different degrees of BBB disruption, were utilized. As expected, U-87 MG derived tumors presented a higher degree of damaged integrity of the BBB than U251 derived tumors (Fig. 5b). Both, NbA42 and NbA213, detected either both U-87 MG and U251 orthotopic brain tumors (Fig. 5c) suggesting that these nanobodies does not present a high dependence for a highly disrupted BBB. Altogether, these results point to NbA42 and NbA213 as candidates for further development of immunotargeted applications for glioblastoma diagnosis and/or therapy.

Discussion
"Omic" sciences hold the promise of precision medicine in cancer by translating molecular-based big data into novel clinical approaches 39 . Performing agnostic bioinformatic analysis across multiple datasets we identified ABCC3 as a suitable candidate to develop immunotargeted tools for glioblastoma. ABCC3 transporter is a member of the multidrug resistance proteins (MRPs) which confers resistance to various chemotherapeutic anticancer compounds 19 . In agreement with other reports we found that ABCC3 is highly expressed in glioblastoma samples, correlates with tumor grade and worse prognosis of patients 23,24 .
ABCC3 may have a plausible role in temozolomide (TMZ) resistance. The expression of ABCC3 increases following TMZ treatment in glioblastoma cell lines, in a concentration and time-dependent manner 40,41 . Also, the specific inhibition of ABCC3 with MK571 increases apoptosis upon TMZ treatment in other cell types 42 . The higher expression of ABCC3 in NK cells modulates their chemoresistance to TMZ in patients of malignant gliomas 43 . Recently, ABCC3 has been included in gene signatures (ABCC3, CD44, TNFRSF1A, and MGMT 24 ; ABCC3, SMC4, EMP3, WEE1, and HIST1H2BK 44 ) that classify glioma patients in agreement with therapeutic response to chemotherapeutic agents, including TMZ. According to previous reports 20 , we found higher ABCC3 expression in patients with methylated MGMT promoter, correlating with a worse prognosis. A role of ABCC3 in cancer stemness is emerging, as ABCC3 knockdown reduced the expression of stemness genes and the CD44 high /CD24 low breast cancer stem-like subpopulation. Also, SOX2 mediates transcription of ABCC3 in www.nature.com/scientificreports/ glioblastoma cells 45 . We found a similar correlation between CD44, FUT4 (CD15) and ABCC3 expression across glioblastoma patient datasets. These data support the possible intervention of ABCC3 in TMZ resistance and cancer stemness. Then, ABCC3 could represent a new molecular target of glioblastoma which may facilitate the subsequent development of molecular imaging approaches for patient management. Novel immunotargeted applications obtained by combining the selectivity of antibody derivatives with the resolution capabilities of a given imaging modality, have been implemented for glioblastoma theragnosis. Singlechain variable fragments (scFvs) against ABCC3 have been used in preclinical models of glioblastoma. Interestingly, scFvs M25, M58, and M89 recognized extracellular epitopes of the N-terminus of ABCC3 and showed specific targeting of ABCC3-expressing glioblastoma cells in vitro 46 . However, the access of scFvs into the CNS is limited and fusion to cell-penetrating peptides acting as BBB shuttles is often required 47 . Nanobodies can circumvent the BBB by taking advantage of molecular routes of transcytosis 15,16 . In recent years, several nanobodies have sprouted against glioblastoma targets of protein biosynthesis (TUFM, TRIM28), DNA metabolism (NAP1L1), and cellular growth (EGFR, DPYSL2, β-Actin) [48][49][50] . Here, we describe two novel nanobodies targeting ABCC3, NbA42 and NbA213, isolated from a previously constructed glioblastoma-specific phage-display library 25 . The rational design of immunogenic-like peptides located on the extracellular surface of ABCC3 allowed a peptide-based strategy for biopanning. In vitro studies showed that NbA42 and NbA213 specifically target ABCC3 at the N-terminus (peptide ABCC3-A). NbA42 and NbA213 recognized ABCC3-expressing cells in glioblastoma heterotopic xenografted tumor-bearing mice. Detection of ABCC3 by NbA42 and NbA213 was further achieved in two orthotopic glioblastoma mouse models with different degrees of BBB disruption. While www.nature.com/scientificreports/ further characterization of the BBB penetrance is required, these results suggest that these nanobodies do not present a high dependence on a highly disrupted BBB. Future applications of both nanobodies targeting ABCC3 may allow the development of novel glioblastoma theragnostic procedures. Nanobodies constitute a versatile tool for cancer management 51 . Among others, nanobodies have been exploited to deliver cytotoxic payloads specifically to tumors, image-guided surgery, and to improve chimeric antigen receptor (CAR)-T and photodynamic therapies [52][53][54][55][56] . Since the approval of nanobody ALX-0681 (Caplacizumab) by the regulatory agencies 57 , examples of radiolabeled nanobodies conducting immuno-positron emission tomography (immuno-PET) 58 studies have reached clinical trials in cancer patients. Regarding glioblastoma, 89 Zr-labeled nanobodies targeting HGF have demonstrated their theragnostic potential in preclinical models 59 . We highlight the potential of NbA42 and NbA213 as new candidate molecules for the further design of nanobody-based molecular imaging probes. The predictive role of ABCC3 in the survival and prognosis of glioblastoma patients reveals a newfangled translational diagnostic and/or therapeutic relevance of NbA42 and NbA213 in the clinics.

Materials and methods
Cell cultures. Human lung adenocarcinoma A549 and its ABCC3 knocked-out by CRISPR/Cas9 system derivative A549 ABCC3KO cell lines, and U-87 MG, U251, U373-MG, and T98G human glioma cell lines were grown with DMEM medium (Sigma-Aldrich) containing 10% FBS (Fisher Scientific), and maintained at 37 °C and 5% CO 2 . A549 and U373-MG were kindly provided by Dr. Martín-Duque (IACS). U-87 MG, U251, and T98G were kindly provided by the Holland and Joyce laboratories (MSKCC). Short tandem repeat markers performed cell line authentication at different passages.
Bioinformatic analysis and statistics. RNA-Seq data from 142 glioblastoma samples and 5 normal brain samples deposited in The Cancer Genome Atlas (TCGA; https:// www. cancer. gov/ tcga accessed on April 26th, 2017) 26 were analyzed using limma (v.3.24.15, R package) 27 to identify the most highly expressed genes. The differentially expressed list of genes was mined for those that encode proteins located in the plasma mem- www.nature.com/scientificreports/ brane. The subcellular localization was determined by experimental immunohistochemistry evidence as cataloged in the Human Protein Atlas (http:// www. prote inatl as. org 35 , "compartments" database 60 ). Gene expression and survival of adult glioblastoma patient data from the TCGA Project (TCGA-GBM and TCGA-GBMLGG), the Chinese Glioma Genome Atlas (CGGA, http:// www. cgga. org. cn/ accessed on October 5th, 2021) 61 , and the Repository for Molecular Brain Neoplasia Data (REMBRANDT, https:// wiki. cance rimag ingar chive. net/ displ ay/ Public/ REMBR ANDT) 62 were analyzed using GlioVis data portal (http:// gliov is. bioin fo. cnio. es/) 28 . Log-rank and Wilcoxon tests were applied for Kaplan-Meier survival curves. Pearson's product-moment correlation was applied to determine gene correlations between paired samples. Tukey's Honest Significant Difference (HSD) was applied to compare ABCC3 expression across tumor grades and glioblastoma vs. normal samples. Statistical analysis was obtained from GlioVis and completed using R (http:// www.r-proje ct. org).

Biopanning of nanobodies. Prediction of immunogenic epitopes of human ABCC3 (UniProtKB ref-
erence: O15438) was performed by submitting the amino acidic sequence to BepiPred-2.0 analysis software (IEDB, https:// www. iedb. org/ accessed on August 1st, 2018) 31 . A list of 57 potential epitopes was obtained (Supplementary Table S2) and manually curated for those located in the outer part of the plasma membrane. Three peptides, corresponding to immunogenic-like extracellular ABCC3 epitopes, were selected and His-tagged synthesized (Thermo Scientific): • ABCC3-A (ALCGSGELGSKFWDSNLSVHTENPDLTPCFHHHHHH), • ABCC3-K (KAEGEISDPFRHHHHHH), • ABCC3-L (HHHHHHSAWTNDAMADSRQNNTSL). www.nature.com/scientificreports/ These peptides were used for biopanning as described elsewhere 63 , using an existing phage-display library of nanobodies generated upon immunization of an adult male alpaca with whole patient-derived stem-like-enriched glioblastoma cells 25 . For each ABCC3-derived peptide, the library was subjected to 3 sequential rounds of biopanning by facing 1 µg peptide in 0.1 M NaHCO 3 , pre-coated on NUNC anti-histidine ELISA plates (Thermo Scientific), with 10 11 phages for 1 h at room temperature. Non-specific binding sites were blocked with 5% nonfat milk. Bound phages were eluted with 100 mM triethylamine and neutralized with 1 M Tris-HCl (pH 7.4). Phages were used to infect the amber-codon-suppressing TG1 E. coli cells for 30 min at 37 °C, which were later co-infected with 10 15 M13K07 helper phages. Enriched phages were grown overnight at 37 °C in 2× Tryptone & Yeast extract medium supplemented with 100 µg/mL ampicillin and 70 µg/mL kanamycin. New produced phages were precipitated with ice-cold polyethylene glycol-6000/NaCl and resuspended in 1 mL PBS.

Production of nanobodies.
Nanobodies with complete sequences were recloned from pHEN4 12 into the pHEN6 64 expression vector, to replace their HA-tag by a hexahistidine(His)-tag for further purification. Briefly, similar colony PCR with primers A6E (5'-GAT GTG CAG CTG CAG GAG TCT GGA GGAGG-3') and 38 (5'-GGA CTA GTG CGG CCG CTG GAG ACG GTG ACC TGG GT-3') yielded products of 400 bp, further cloned into pHEN6 with PstI and BstEII (Eco91I) (Thermo Scientific). Conservation of sequences was verified by PCR with FP (5'-CGC CAG GGT TTT CCC AGT CAC GAC -3') and RP (5'-TCA CAC AGG AAA CAG CTA TGAC-3'), with further sequencing. Expression of nanobodies encoded in the pHEN6 vector was performed in WK6 E. coli as previously described 64 . Briefly, 1 mM of IPTG was added to cultures to induce nanobody expression overnight at 28 ˚C. Periplasmic extracts were obtained by osmotic shock and dialyzed on Spectra/Por dialysis membrane tubing of MWCO 3.5 kDa (Spectrum laboratories, Inc) in PBS. His-tagged nanobodies were purified using an increasing gradient of 0.5 M imidazole on immobilized metal affinity chromatography columns in an ÄKTA protein purification system (GE Healthcare). Nanobody concentration was measured on a Synergy HT multimode microplate reader (BioTek) after incubation with Bradford reagent (Bio-Rad Laboratories, Inc).

Animal ethics statement and experimental design. Athymic
For orthotopic xenografts, U-87 MG and 251 cells were transduced with a TK-GFP-Luciferase reporter plasmid (TGL) to monitor tumor growth by bioluminescence measurements. To develop intracranial tumors, 6-8 weeks athymic nude mice were anesthetized by inhalation of 3% isoflurane and were subcutaneously injected with 50 μL of the local anesthetic 0.25% bupivacaine at the surgical site. Mice were intracranially injected with 2 μL containing 5·10 5 U-87 MG-TGL or 251-MG-TGL cells resuspended in PBS, using a fixed stereotactic apparatus (Stoelting). Injections were made to the right frontal cortex, approximately 1 mm caudal and 1.5 mm lateral www.nature.com/scientificreports/ from bregma, and at a depth of 2 mm using a Hamilton syringe (Hamilton) as described previously 65,66 . Tumor growth was monitored by bioluminescence. Mice were injected with D-Luciferin (150 mg/kg). 15 min after injection, images were acquired for 5 s using an IVIS Lumina XRMS equipment (Perkin Elmer). Bioluminescence analysis was performed using Living Image software, version 2.50. Nanobodies were administered as described above in mice with tumors in a positive growth phase determined by bioluminescence (10 6 to 10 8 p/s/cm 2 /sr of average radiance). Mice were sacrificed 1 h after administration, and tumors were processed as described above.
To evaluate the BBB disruption, 6-8 weeks athymic nude littermates bearing similar intracranial tumors were injected i.p. with 400 μL of 4% Evan's blue dye (Sigma). One hour after injection, mice were sacrificed by CO 2 inhalation and perfused with acidified fixative (1% PFA in 0.05 mM citrate buffer, pH 3.5). Collected brain tumors and samples of their contralateral hemisphere were incubated in 700 μL of formamide (Sigma) to extract Evan's blue at 60 °C overnight. Absorbance was measured at 610 nm and 740 nm on a Synergy HT multi-mode microplate reader (BioTek). Non-injected or intracranially injected with 2 μL of PBS (mock surgery), 6-8 weeks athymic nude littermates were included as intact brain controls.
To evaluate the bioavailability of nanobodies plasma and urine samples were obtained from mice sacrificed 15 min, 30 min, 1 h, 4 h, and 24 h after administration of nanobodies. Protein content was measured in plasma and urine samples using a Synergy HT multi-mode microplate reader (BioTek) after incubation with Bradford reagent (Bio-Rad Laboratories, Inc). A volume containing 5 μg of protein for plasma and 1 μg for urine was used for immunoblot detection. ReadyTag anti-6-His mAb (1:1,000; clone 6-HIS, BioXCell) and goat anti-mouse IgG IRDye800CW antibody (1:20,000; LI-COR) allowed detection of His-tagged nanobodies in an Odyssey CLx Imaging System (LI-COR). Vehicle-treated healthy mice were used as controls.
Experimental statement. All experiments and methods were performed in accordance with relevant guidelines and regulations.

Data availability
Data supporting the findings of this manuscript are available from the corresponding author upon reasonable request. Data for bioinformatic analysis are deposited in publicly available patient datasets (e.g. RNA-Seq data from The Cancer Genome Atlas (TCGA; https:// www. cancer. gov/ tcga); gene expression and patient survival data from TCGA Project (TCGA-GBM and TCGA-GBMLGG), the Chinese Glioma Genome Atlas (CGGA, http:// www. cgga. org. cn), and the Repository for Molecular Brain Neoplasia Data (REMBRANDT, https:// wiki. cance rimag ingar chive. net/ displ ay/ Public/ REMBR ANDT).